Optical fiber biosensor probe and method of making same

Abstract

The optical fiber biosensor probe and the preparation method thereof provided by the present disclosure, the optical fiber biosensor probe comprises: a conductive optical fiber; sensing cells capable of expressing fluorescent indicator proteins located at or near the end of the conductive optical fiber, and the sensing cells are dispersed in a hydrogel-based cell culture environment; and a space environment connected with the conductive optical fiber for providing protection for the sensing cells. The preparation method comprises: preparing sensing cells capable of expressing target fluorescent indicator proteins through cell transfection; transferring the sensing cells into a hydrogel-based cell culture environment to obtain hydrogel containing sensing cells; assembling the conductive optical fiber to the space environment, transferring the hydrogel containing sensing cells to the space environment at or near the end of the conductive optical fiber, and obtaining the optical fiber biosensor probe after the hydrogel solidifies. The optical fiber biosensor probe provided by the present disclosure can realize real-time monitoring with high chemical specificity.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of optical fiber sensing, and particularly relates to an optical fiber biosensor probe and a preparation method thereof. BACKGROUND

[0002] With the continuous development of biological detection technology, human health monitoring and disease diagnosis level has also been improved by leaps and bounds. However, the current biomedical detection technology still cannot meet the application requirements of high detection accuracy, high specificity and high time resolution. For example, in the field of brain science research, brain disease detection, cancer detection, etc., there are still great challenges in rapid quantitative detection and in vivo real-time monitoring of some kinds of complex and similar biological molecules (such as neurotransmitters).

[0003] Common biological molecule detection technologies, such as electrochemical method, high performance liquid chromatography, colorimetric method, fluorescence spectroscopy, etc., cannot meet the detection requirements of high time resolution and high chemical specificity. Electrochemical method has the advantages of fast speed and high sensitivity, but it is difficult to distinguish similar potential biological molecules. Moreover, the electrochemical method used for in vivo detection needs to overcome the immune response, which further increases the difficulty of electrode manufacturing. High performance liquid chromatography has good selectivity, but the detection time is long, the equipment is expensive and complex, and it does not have the ability of in vivo real-time monitoring. Colorimetric method and fluorescence spectroscopy have the advantages of simplicity and high sensitivity, but they need to pretreat the detection sample, which is not suitable for in vivo detection.

[0004] Due to the advantages of low loss, small size, low cost and anti-electromagnetic interference of optical fiber, optical fiber fluorescence sensor has been widely applied and developed in the fields of biological medicine and in vivo detection. In the optical fiber fluorescence sensor, the sensitive performance of the fluorescent material determines the performance (such as sensitivity) of the optical fiber fluorescence sensor. Common fluorescent materials are divided into inorganic fluorescent materials and organic fluorescent materials. Among them, the representative of inorganic fluorescent materials is rare earth ion luminescence and rare earth fluorescent materials; organic fluorescent materials can be divided into organic small molecule fluorescent materials, organic polymer fluorescent materials, organic complex fluorescent materials, etc. When it is applied to biochemical sensing, the basic principle of the optical fiber fluorescence sensor is to detect the changes of fluorescence intensity, spectrum or lifetime caused by the combination of the measured substance and the fluorescent material or the change of the measured physiological parameter. The above process usually has low chemical specificity, which is difficult to meet the application requirements of high specificity detection.

[0005] In order to achieve high time resolution and high chemical specificity in vivo detection of biochemical substances, a series of genetically encoded fluorescent protein probes have been developed by using genetic engineering technology, and real-time monitoring is carried out by combining fluorescence microscopy or fiber optic photometer. Among them, expressing fluorescent protein probes in the target area is a time-consuming and laborious work, and there are currently two ways to cultivate transgenic animal models and viral transfection. The former cultivation period is related to the breeding period of the model animal, and generally takes a long time; the latter can shorten the expression time of the fluorescent protein probe to about 2 weeks, but it still cannot achieve real-time detection, and its transfection efficiency depends on the selected promoter gene, and heavy work is needed to explore the appropriate conditions when used in different model animals.

[0006] In summary, how to realize real-time sensing with high time resolution and high chemical specificity is a technical difficulty that needs to be overcome in the field of optical fiber sensing. SUMMARY

[0007] The present disclosure aims to at least partially solve one of the technical problems in the related art.

[0008] To this end, one object of the present disclosure is to provide an optical fiber biosensor probe with high time resolution and high chemical specificity. The sensing probe assembles sensing cells that can express fluorescent indicator proteins (also known as fluorescent protein probes) into the microchannels at the end face of the optical fiber or near the end face of the optical fiber, which can realize real-time monitoring with high specificity and has broad application prospects in the field of biomedicine. The optical fiber biosensor probe provided by the first aspect of the present disclosure comprises:

[0009] a conductive optical fiber;

[0010] sensing cells that can express fluorescent indicator proteins located at the end or near the end of the conductive optical fiber, and the sensing cells are dispersed in a cell culture environment based on hydrogel; and

[0011] a spatial environment connected to the conductive optical fiber for providing protection for the sensing cells.

[0012] The optical fiber biosensor probe provided by the first aspect of the present disclosure has the following advantages compared with the prior art:

[0013] (1) High time resolution and high chemical specificity. The sensing probe is based on the sensing of fluorescent indicator proteins expressed by sensing cells, and has the advantages of high chemical specificity and fast response time.

[0014] (2) Plug and play. Compared with the conventional method of first in vivo viral transfection and then waiting for several weeks for sensing or first cultivating transgenic model organisms and then sensing, the sensing probe can realize plug and play without waiting time.

[0015] (3) Avoid immune response. In the sensor probe, the sensing cells are wrapped by hydrogel, and the sensing cells do not contact with the tissue cells in the detection area, so that the immune rejection reaction of the tissue to the foreign cells in the in vivo detection is avoided.

[0016] (4) Universality. In the sensor probe, the fluorescent indicator protein is expressed in the sensing cells, and the problems of the promoter gene required for the expression of the fluorescent indicator protein in different animal models do not need to be considered, and the sensor probe is not dependent on the type of target animal model, and has universality.

[0017] (5) Small invasiveness, suitable for integration with electrodes, surgical needles, and endoscopy systems. The sensor uses an optical fiber or a capillary glass tube as a sensor probe for in vivo detection, has the advantages of small size and small damage to the biological brain tissue, and is suitable for integration with electrodes, surgical needles, and endoscopy systems, and can realize multi-modal and multi-parameter detection.

[0018] In summary, the optical fiber biosensor probe has the advantages of high time resolution and high chemical specificity, and can be used for biochemical substance sensing and physiological parameter monitoring.

[0019] In some embodiments, the sensing cells are genetically engineered and obtained by cell transfection, and can express the target fluorescent indicator protein.

[0020] In some embodiments, the cell line is selected from a human embryonic kidney cell line HEK293, a human cervical cancer cell line HeLa, a mouse lymphoma cell line EL4, a human bone marrow mesenchymal stem cell line hMSC, or a human lung cancer cell line Hep3B.

[0021] In some embodiments, the cell transfection is performed by transient transfection or stable transfection.

[0022] In some embodiments, the sensing cells can express at least one fluorescent indicator protein sensitive to the biochemical substance or physiological parameter to be detected.

[0023] In some embodiments, the sensing cells can express a fluorescent protein having a different emission spectrum than the selected fluorescent indicator protein and not having a probe attribute.

[0024] In some embodiments, the density of the sensing cells in the cell culture environment is 10 2 to 10 9 per mL.

[0025] In some embodiments, the spatial environment is a micro-sleeve, the conductive optical fiber is fixed in the micro-sleeve, and an electrode is arranged side by side with the conductive optical fiber in the micro-sleeve.

[0026] In some embodiments, the spatial environment is a microchannel of an optical fiber, and the microchannel is a groove or a through-hole with an arbitrary shape fabricated by microfabrication near the end face of the optical fiber.

[0027] The second aspect of the present disclosure provides a preparation method of the optical fiber biosensor probe, wherein the optical fiber biosensor probe is the optical fiber biosensor probe according to the first aspect of the present disclosure, and the preparation method comprises:

[0028] The sensing cells capable of expressing the target fluorescent indicator protein are prepared by cell transfection;

[0029] The sensing cells are transferred into a hydrogel-based cell culture environment to obtain a hydrogel containing the sensing cells;

[0030] The optical fiber is assembled into the spatial environment, and the hydrogel containing the sensing cells is transferred into the end or the vicinity of the end of the optical fiber in the spatial environment, and the optical fiber biosensor probe is obtained after the hydrogel is solidified. BRIEF DESCRIPTION OF DRAWINGS

[0031] The above-mentioned and / or additional aspects and advantages of the present disclosure will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings, in which:

[0032] Figure 1 FIG. 1 is a structural schematic diagram of an optical fiber biosensor probe according to an embodiment of the present disclosure.

[0033] Figure 2 FIG. 2 is another structural schematic diagram of an optical fiber biosensor probe according to an embodiment of the present disclosure.

[0034] Figure 3 FIG. 3 is a manufacturing step of an optical fiber biosensor probe according to an embodiment of the present disclosure.

[0035] Figure 4 FIG. 4 is another manufacturing step of an optical fiber biosensor probe according to an embodiment of the present disclosure.

[0036] Figure 5 FIG. 5 is a manufacturing step of an optical fiber biosensor probe according to another embodiment of the present disclosure.

[0037] Figure 6 FIG. 6 is a structural diagram of a neurotransmitter dynamic detection system of an optical fiber biosensor probe according to an embodiment of the present disclosure.

[0038] Figure 7 FIG. 7 is a fluorescent signal obtained by detecting norepinephrine by an optical fiber biosensor probe according to an embodiment of the present disclosure.

[0039] Figure 8Fluorescence microscopic images of a fiber-optic biosensor probe according to one embodiment of the present disclosure before and after being placed in a noradrenaline solution.

[0040] Figure 9 Dynamic changes in noradrenaline concentration observed after the fiber-optic biosensor probe according to one embodiment of the present disclosure was placed behind the hypothalamus of a mouse and the mouse was forced to swim.

[0041] Reference Signs:

[0042] 1 - spatial environment, 2 - conducting optical fiber, 3 - cell culture environment, 4 - hydrogel, 5 - sensing cell. DETAILED DESCRIPTION

[0043] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and should not be used to limit the present application.

[0044] On the contrary, the present application covers any alternatives, modifications, equivalent methods and solutions defined by the claims within the spirit and scope of the present application. Further, in order to make the public better understand the present application, some specific details are described in the following detailed description of the present application. The present application can also be completely understood without the description of these details by those skilled in the art.

[0045] Reference Signs: Figure 1 A structural schematic diagram of a fiber-optic biosensor probe according to one embodiment of the present disclosure. The fiber-optic biosensor probe comprises:

[0046] a conducting optical fiber 2;

[0047] sensing cells 5 capable of expressing fluorescent indicator proteins located at or near the end of the conducting optical fiber 2, and the sensing cells 5 are dispersed in a cell culture environment 3 based on a hydrogel 4; and

[0048] a spatial environment 1 connected to the conducting optical fiber 2 for providing protection for the sensing cells 5.

[0049] Further, the sensing cells 5 in the fiber-optic biosensor probe are cell lines modified by genetic engineering technology and capable of expressing fluorescent indicator proteins, such as those obtained by cell transfection in human embryonic kidney cell line HEK293, human cervical cancer cell line HeLa, mouse lymphoma cell line EL4, human bone marrow mesenchymal stem cell line hMSC, human lung cancer cell line Hep3B, etc.

[0050] Preferably, the cell transfection method includes transient transfection or stable transfection, and the cell transfection method adopts physical mediation (electroporation, gene gun method, microinjection), chemical mediation (liposome transfection, calcium phosphate coprecipitation, cationic polymer mediation) or biological mediation (virus-mediated transfection, protoplast transfection) and the like.

[0051] Preferably, the sensing cells 5 in the optical fiber biosensor probe can express fluorescent indicator proteins sensitive to at least one biochemical substance or physiological parameter to be measured, such as genetically encoded fluorescent probes based on G protein-coupled receptors (GPCRs). If there are multiple substances to be measured, several corresponding fluorescent indicator proteins with different emission spectra or fluorescence lifetimes are used to distinguish different substances to be measured.

[0052] Preferably, the sensing cells 5 in the optical fiber biosensor probe can additionally express fluorescent proteins with different emission spectra than the selected fluorescent indicator proteins, such as green fluorescent protein GFP and red fluorescent protein RFP, which do not have probe properties, to calibrate the loss of the sensing system, eliminate signal interference caused by environmental disturbances or animal movement, and achieve robust measurement.

[0053] Preferably, the cell culture environment 3 based on hydrogel 4 in the optical fiber biosensor probe can use PEG (polyethylene glycol) derivative hydrogel, sodium alginate hydrogel or Matrigel.

[0054] Preferably, the density of the sensing cells 5 in the cell culture environment 3 in the optical fiber biosensor probe can be 10 2 ~ 10 9 cells / mL.

[0055] Preferably, the conducting optical fiber 2 in the optical fiber biosensor probe can be a high numerical aperture multimode optical fiber, a photonic crystal optical fiber or a microstructured optical fiber.

[0056] Preferably, the spatial environment 1 for providing cell protection in the optical fiber biosensor probe is a micro-sleeve, the inner diameter of the micro-sleeve is slightly larger than the diameter of the conducting optical fiber 2, and the material can be glass, rubber or plastic. The conducting optical fiber 2 should be fixed in the micro-sleeve, and the end surface of the micro-sleeve should be slightly convex relative to the end surface of the conducting optical fiber 2 by 0.01-3 mm to accommodate the sensing cells 5.

[0057] Preferably, when the spatial environment 1 for providing cell protection in the optical fiber biosensor probe is a micro-sleeve, a metal electrode can be assembled with the conducting optical fiber 2 to perform synchronous local field potential monitoring. The metal electrode is fixed side by side with the conducting optical fiber 2 in the micro-sleeve, and the end surface of the metal electrode is about 0.1 mm-2 mm relative to the end surface of the micro-sleeve.

[0058] The working principle of the optical fiber biosensor probe provided by the embodiments of the present disclosure is as follows:

[0059] The conducting optical fiber 2 conducts excitation light corresponding to the fluorescent indicator protein expressed by the sensing cell 5 to the end or the vicinity of the end of the conducting optical fiber; when the fluorescent indicator protein expressed by the sensing cell 5 binds to the to-be-detected substance released in the biological tissue, the fluorescence signal (such as intensity, spectrum, lifetime, etc.) changes; the conducting optical fiber 2 collects the fluorescence signal and conducts it to the detection system. The cell culture environment 3 based on the hydrogel 4 provides a growth environment for the sensing cell 5; the spatial environment 1 provides protection for the installation of the sensing probe and prevents the surrounding tissue from being extruded during the installation of the sensing probe.

[0060] Referring to Figure 2 , another structural schematic diagram of the optical fiber biosensor probe of one embodiment of the present disclosure, the optical fiber biosensor probe of the present embodiment is different from the optical fiber biosensor probe of the embodiment shown in Figure 1 . In the present embodiment, the spatial environment 1 for providing cell protection is a fiber microchannel. The fiber microchannel can be a groove or a through hole with an arbitrary shape made by microfabrication near the end face of the conducting optical fiber 2.

[0061] The present disclosure further provides a preparation method of the above-mentioned optical fiber biosensor probe, comprising the following steps:

[0062] Step (1) preparing sensing cells capable of expressing target fluorescent indicator proteins by cell transfection

[0063] Step (2) transferring the sensing cells to a three-dimensional cell culture medium of a hydrogel. The sensing cells after transfection are digested to obtain a cell suspension, and a high-concentration cell suspension (10 2 ~ 10 9 mL) is obtained by centrifugation; then the high-concentration cell suspension is mixed with the hydrogel at a volume ratio of 1:20 to 20:1 to obtain a hydrogel containing sensing cells.

[0064] Step (3) assembling the conducting optical fiber to the spatial environment and transferring the hydrogel containing the sensing cells to the end or the vicinity of the end of the conducting optical fiber in the spatial environment, specifically including: using a laser microfabrication method to make a rectangular window at one end of a micro sleeve and fixing the conducting optical fiber in the micro sleeve (the end face of the optical fiber is in the rectangular window), or using a laser microfabrication method to make a groove or a through hole with an arbitrary shape near the end face of the conducting optical fiber, and then transferring the hydrogel containing the sensing cells obtained in step (2) to the vicinity of the end face of the conducting optical fiber in the micro sleeve or the groove or the through hole obtained by microfabrication in the conducting optical fiber. After the hydrogel is solidified, the sensing probe is completed.

[0065] Embodiment one of the method for preparing the optical fiber biosensor probe of the present disclosure is as follows, referring to Figure 3 The specific preparation process is as follows: step (1) making a sensing cell that can express a fluorescent probe by cell transfection, specifically including:

[0066] For example, take one of the transient transfection methods, liposome transfection, mix the sensing cell to be transfected, plasmid containing the target fluorescent probe gene, liposome, culture medium, etc., and co-culture in a culture dish. Replace the culture medium after 6 hours, and generally obtain the genetically modified sensing cell that can express the fluorescent probe after 24 hours. For example, mix the sensing cell HEK293, plasmid containing the target fluorescent probe gene (such as norepinephrine probe plasmid, reference Neuron 102, 745-761, 2019), liposome, and culture medium (such as high-sugar DMEM (Dulbecco's Modified Eagle Medium) + 10% v / v FBS (Fetal Bovine Serum)) in a culture dish. When the cell density reaches 60%, add the plasmid and liposome to the culture medium (Gibco DMEM, serum-free) at a volume ratio of 1:3. For example, take a 100 mm diameter culture dish, take 15 μl plasmid and 45 μl liposome, and add them to 800 μL culture medium (Gibco DMEM, serum-free). Shake gently and let stand for 5 minutes. Mix the plasmid solution and liposome solution and let stand for 20 minutes. Slowly drop the mixed solution into the culture dish drop by drop. Replace the culture medium (DMEM + 10% v / v FBS + 1% v / v antibiotic P / S (Penicillin-Streptomycin Solution)) about 6 hours after transfection, and generally obtain the genetically modified sensing cell that can express the target fluorescent probe after 24 hours.

[0067] Step (2) transferring the sensing cell to the hydrogel three-dimensional cell culture medium, specifically including:

[0068] The sensing cell after transfection for about 24 hours is digested with trypsin to obtain a cell suspension, and then high-concentration cell suspension (~ 10 7The cell suspension and the hydrogel (e.g., Matrigel) are mixed at a volume ratio of 1:3 to obtain a hydrogel containing the sensing cells. In addition, an equal volume of culture medium (DMEM+10% FBS) can be added to the mixed hydrogel containing the sensing cells, and the mixture is placed in a cell incubator at 37°C and a volume concentration of 5% CO2 for 30 minutes (the solidification time is related to the volume of the hydrogel, and the optimal solidification time for a 500-microliter hydrogel is 30 minutes). Then, the hydrogel containing the sensing cells that has been solidified is taken out, and the excess culture medium on the surface is removed and replaced with new culture medium.

[0069] Step (3) assembling the hydrogel containing the sensing cells to the micro-cannula assembly, specifically including:

[0070] The micro-cannula is fabricated on the capillary glass tube by laser microfabrication. The size of the capillary glass tube can be determined according to the size of the optical fiber and different biological detection requirements. For example, a common cylindrical glass tube BJ-40 (inner diameter 0.8 mm, outer diameter 1 mm) is cut at a distance of about 1 mm from one end of the capillary glass tube and about 0.24 mm from the center line of the capillary glass tube to form a rectangular window (length about 1.5 mm). The end of the conductive optical fiber is fixed near the rectangular window of the capillary glass tube by ultraviolet glue (the end face of the capillary glass tube protrudes about 0.5 mm relative to the end face of the conductive optical fiber), and then the hydrogel containing the sensing cells obtained in step (2) is transferred to the end face of the optical fiber in the micro-cannula. After being placed in a cell incubator for 2-5 minutes (since the volume of the single probe hydrogel is about 20 nanoliters, the optimal solidification time is 4 minutes), the Matrigel hydrogel is solidified, and the sensing probe is completed.

[0071] In other embodiments, the culture medium used in step (2) can be BME, MEM, HAM F12, PRMI1640, M199, IMDM, L15, etc.

[0072] The following is Example Two of the method for preparing the optical fiber biosensing probe according to the present disclosure, which is described with reference to Figure 4 , and the specific preparation process is as follows: Step (1) preparing sensing cells capable of expressing fluorescent protein probes by cell transfection, specifically including:

[0073] Take one of the instant transfection methods-liposome transfection as an example, the cells to be transfected, plasmids containing target fluorescent probe genes, liposomes, culture medium, etc. are mixed and cultured in a culture dish. After 6 hours, the culture medium is replaced, and generally after 24 hours, the genetically modified cells that can express fluorescent probes are obtained. For example, the sensing cells HeLa, plasmids containing target fluorescent probe genes (such as adenosine probe plasmid, reference https: / / doi.org / 10.1101 / 2020.05.04.075564) and liposomes, culture medium (such as DMEM+10% v / v FBS) are mixed and cultured in a culture dish. When the cell density reaches 50-80%, the plasmid and liposome are added to the culture medium (Gibco DMEM, no serum) at a volume ratio of 1:2. For example, take a 60 mm diameter culture dish, take 20 μl plasmid and 40 μl liposome, add 400 μL culture medium (Gibco DMEM, no serum), shake gently and stand for 5 minutes. Mix the plasmid solution and liposome solution, and stand for 20 minutes. Slowly drop the mixed solution into the culture dish drop by drop. Replace the culture medium (DMEM+10% v / v FBS+1% v / v P / S) about 6 hours after transfection, and generally after 24 hours, the genetically modified cells that can express fluorescent probes are obtained.

[0074] Step (2) transferring the sensing cells to the hydrogel three-dimensional cell culture medium, specifically including:

[0075] The sensing cells after transfection for about 24 hours are digested with trypsin to obtain a cell suspension, and then a high-concentration cell suspension (10 2 ~10 9 cells / mL) is obtained by centrifugation (speed 1000 rpm, temperature 20°C, time 5 min); mix the cell suspension and hydrogel (such as Matrigel) at a volume ratio of 1:1 to obtain a hydrogel containing cells.

[0076] Step (3) assembling the hydrogel containing sensing cells to the microcannula assembly, specifically including:

[0077] The micro-cannula is made on the capillary optical fiber by laser micro-processing. The size of the capillary optical fiber can be determined according to the size of the optical fiber and different biological detection requirements. Take the capillary optical fiber HCCF_PSC320 / 340 / 360PI (inner diameter 320 microns, outer diameter 410 microns, plus coating layer 440 microns, http: / / www.microphotons.cn / index.php?a=cpinfo&id=884) suitable for in vivo detection of mice as an example. A rectangular window (length 0.5-3 mm) can be cut at a distance of 0.5-3 mm from one end of the capillary optical fiber and at a distance of 0.16 mm from the center line of the capillary optical fiber. The conductive optical fiber and the metal electrode are fixed near the rectangular window of the capillary glass tube by UV glue (the end face of the capillary glass tube is slightly convex to the end face of the conductive optical fiber by 0.01-3 mm, and the metal electrode protrudes from the end face of the capillary glass tube by 0-300 μm). Then the hydrogel containing cells obtained in step (2) is transferred to the end face of the optical fiber in the micro-cannula. Place in a 37°C, 5% cell incubator for 1 min, and the Matrigel hydrogel is solidified. The sensing probe is completed. In order to be preserved and used for a long time, the sensing probe is fixed at the bottom of the culture dish. Take a culture dish with a diameter of 60 mm as an example, add 3 mL of culture medium (DMEM+10% v / v FBS+1% v / v P / S), and place it in a 37°C, 5% cell incubator. The sensing probe is preserved in the cell incubator for 0-6 hours, and the sensing effect is basically unchanged.

[0078] The following is Example Three of the method for preparing the optical fiber biological sensing probe of the present disclosure, which is described with reference to Figure 6 , and the specific preparation process is as follows:

[0079] Step (1) Prepare sensing cells that can express fluorescent protein probes by cell transfection. Specifically, it includes:

[0080] Take one of the instant transfection methods-liposome transfection as an example, the cells to be transfected, plasmids containing target fluorescent probe genes, liposomes, culture medium, etc. are mixed and cultured in a culture dish. After 6 hours, the culture medium is replaced, and generally after 24 hours, the genetically modified cells that can express fluorescent probes are obtained. For example, the sensing cells Hep3B, plasmids containing target fluorescent probe genes (such as acetylcholine probe plasmid, reference https: / / doi.org / 10.1038 / s41592-020-0953-2) and liposomes, culture medium (such as DMEM+10% v / v FBS) are mixed and cultured in a culture dish. When the cell density reaches 50-80%, the plasmid and liposome are added to the culture medium (Gibco DMEM, no serum) at a volume ratio of 1:1. For example, take a 60 mm diameter culture dish, take 20 μl plasmid and 20 μl liposome, add 400 μL culture medium (Gibco DMEM, no serum), shake gently and stand for 5 minutes. Mix the plasmid solution and liposome solution, and stand for 20 minutes. Slowly drop the mixed solution into the culture dish drop by drop. Replace the culture medium (DMEM+10% v / v FBS+1% P / S) about 6 hours after transfection, and generally after 24 hours, the genetically modified cells that can express fluorescent probes are obtained.

[0081] Step (2) transferring the sensing cells to the hydrogel three-dimensional culture medium, specifically including:

[0082] The sensing cells after transfection for about 24 hours are digested with trypsin to obtain a cell suspension, and then a high concentration of cell suspension (10 2 ~10 9 cells / mL) is obtained by centrifugation (speed 1000 rpm, temperature 20°C, time 5 min); mix the cell suspension and hydrogel (such as Matrigel) at a volume ratio of 1:1 to obtain a hydrogel containing cells.

[0083] Step (3) assembling the hydrogel containing sensing cells to the optical fiber microchannel, specifically including:

[0084] The microchannel is made near the end of the conducting optical fiber by laser microprocessing, for example, a rectangular groove (length 0.2-3 mm, width ~ 60 μm, depth 20-220 μm) is cut at one end of a multimode optical fiber (core 200 μm, cladding 220 μm, NA 0.22) 0.1-3 mm away, and then the hydrogel containing the sensing cells obtained in step (2) is transferred into the microchannel. Place in a 37°C, 5% cell incubator for 1 min, and the Matrigel hydrogel is solidified, and the sensing probe is completed. In order to preserve and use for a long time, the sensing probe is fixed to the bottom of the culture dish, for example, a culture dish with a diameter of 60 mm, 3 mL of culture medium (DMEM + 10% v / v FBS + 1% v / v P / S) is added, and it is placed in a 37°C, 5% cell incubator. The sensing probe is stored in the cell incubator for 0-6 hours, and the sensing effect is basically unchanged.

[0085] The following is an application case of the optical fiber biosensing probe of the present disclosure:

[0086] Referring to Example One of the present disclosure, the plasmid containing norepinephrine sensing protein and red fluorescent protein sequence reported in the literature Feng et al., Neuron 102, 745-761 (2019) is used for cell transfection in HEK293 cell line, to obtain sensing cells expressing norepinephrine fluorescent sensing protein (green) and ordinary red fluorescent protein, and to be placed at the end of the sensing optical fiber, to further make a norepinephrine optical fiber biosensing probe. Figure 6 The figure is a structure diagram of a neurotransmitter dynamic detection system for the optical fiber biosensing probe according to one embodiment of the present disclosure, including a light source, a light splitting system, a conducting optical fiber, an optical fiber biosensing probe, and a detection system, wherein the light splitting system projects the excitation light and the emission fluorescence of different fluorescent proteins to different ports for excitation or detection.

[0087] Figure 7 The system shown in Figure 6 The fluorescence signal detected by placing the norepinephrine optical fiber biosensing probe in a 10 μM norepinephrine solution shows that the norepinephrine sensing signal rises (light curve), while the reference signal in the red channel does not change (dark curve), thus the response time of the norepinephrine optical fiber biosensing probe can be obtained.

[0088] Figure 8 The fluorescence signal detected by placing the norepinephrine optical fiber biosensing probe in different concentrations of norepinephrine solution. It can be seen that the intensity of the fluorescence signal can reflect the concentration of the norepinephrine solution, and this optical fiber biosensing probe can be applied to the concentration detection of norepinephrine.

[0089] Figure 9The fluorescence microscopic images of the noradrenaline optical fiber biosensor probe before and after being placed in 10 μM noradrenaline solution. After the noradrenaline is applied, the noradrenaline sensing signal (green channel, uplink) rises, while the reference signal in the red channel (downlink) does not change, indicating that the probe has the sensing ability. (The leftmost column: before applying noradrenaline, the middle column: after applying noradrenaline, the rightmost column: the amount of change before and after applying noradrenaline)

[0090] Figure 10 The dynamic changes of the noradrenaline sensing signal observed in the forced swimming behavioral paradigm of the noradrenaline optical fiber biosensor probe implanted in the target brain region (here, the hypothalamus) of a model animal (such as a mouse). The mouse is placed in water and forced to swim for three minutes, and the fluorescence signal is observed to increase, indicating an increase in noradrenaline concentration; the mouse is then removed from the water, and the fluorescence signal decreases, indicating a decrease in noradrenaline concentration. The above process is repeated three times, and the noradrenaline signal increases and then decreases, indicating that the probe has good repeatability and can be applied to the field of dynamic detection of noradrenaline concentration.

[0091] In summary, the present disclosure is based on the sensing cells expressing fluorescent protein probes (also known as fluorescent indicator proteins) for detecting biochemical substances and physiological parameters, and has the advantages of high chemical specificity and high time resolution. In addition, the probe also has the unique advantages of plug-and-play, avoiding immune rejection, being applicable to a variety of model animals, having low invasiveness, being easy to combine with medical instruments, etc., and has a wide application prospect in the field of optical fiber sensing.

[0092] The description and application of the present application herein are illustrative and are not intended to limit the scope of the present application to the above embodiments. Variations and changes to the disclosed embodiments are possible, and various components of the embodiments are known to those of ordinary skill in the art. It should be clear to those skilled in the art that the present application can be implemented in other forms, structures, arrangements, proportions, and with other components, materials and parts without departing from the spirit or essential characteristics of the present application. Other variations and changes to the disclosed embodiments can be made without departing from the scope and spirit of the present application.

Claims

1. An optical fiber biosensor probe, characterized by, The application relates to a fiber-optic biosensor probe, comprising: a conductive optical fiber; a sensing cell capable of expressing a fluorescent indicator protein located at or near the end of the conductive optical fiber, and the sensing cell is dispersed in a hydrogel-based cell culture environment; and a space environment connected with the conductive optical fiber for providing protection for the sensing cell; the space environment is a micro-sleeve, the conductive optical fiber is fixed in the micro-sleeve, an electrode is arranged in parallel with the conductive optical fiber in the micro-sleeve, a rectangular window is formed at one end of the micro-sleeve at the end surface of the conductive optical fiber, and the sensing cell is located in the rectangular window; or the space environment is an optical fiber micro-channel, the optical fiber micro-channel is a through hole with an arbitrary shape which is formed by micro-processing near the end surface of the conductive optical fiber, and the sensing cell is located in the through hole. The sensing cell is genetically modified and obtained by cell transfection, and can express a target fluorescent indicator protein. The cell line is selected from a human embryonic kidney cell line HEK293, a human cervical cancer cell line HeLa, a mouse lymphoma cell line EL4, a human bone marrow mesenchymal stem cell line hMSC or a human lung cancer cell line Hep3B. The cell transfection adopts transient transfection or stable transfection. The sensing cell can express a fluorescent indicator protein sensitive to at least one biochemical substance or physiological parameter to be detected. The sensing cell can express a fluorescent protein with a different emission spectrum from the selected fluorescent indicator protein and without a probe attribute. The fiber-optic biosensor probe is prepared according to any one of claims 1-7, and the preparation method comprises: preparing a sensing cell capable of expressing a target fluorescent indicator protein by cell transfection; transferring the sensing cell into a hydrogel-based cell culture environment to obtain a hydrogel containing the sensing cell; assembling the conductive optical fiber into the space environment, transferring the hydrogel containing the sensing cell into the space environment at or near the end of the conductive optical fiber, and obtaining the fiber-optic biosensor probe after the hydrogel is solidified.

2. The optical fiber biosensing probe of claim 1, wherein, ​ 3. The optical fiber biosensing probe of claim 2, wherein, ​ 4. The optical fiber biosensing probe of claim 2, wherein, ​ 5. The optical fiber biosensing probe of claim 1, wherein, ​ 6. The optical fiber biosensing probe of claim 1, wherein, ​ 7. The optical fiber biosensing probe of claim 1, wherein, The density of the induced cells in the cell culture environment is 10 2 to 10 9 cells / mL.

8. A method of making an optical fiber biosensor probe, comprising: ​ ​ ​ ​

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